Steroid-associated hip joint collapse in bipedal emus

In this study we established a bipedal animal model of steroid-associated hip joint collapse in emus for testing potential treatment protocols to be developed for prevention of steroid-associated joint collapse in preclinical settings. Five adult male emus were treated with a steroid-associated osteonecrosis (SAON) induction protocol using combination of pulsed lipopolysaccharide (LPS) and methylprednisolone (MPS). Additional three emus were used as normal control. Post-induction, emu gait was observed, magnetic resonance imaging (MRI) was performed, and blood was collected for routine examination, including testing blood coagulation and lipid metabolism. Emus were sacrificed at week 24 post-induction, bilateral femora were collected for micro-computed tomography (micro-CT) and histological analysis. Asymmetric limping gait and abnormal MRI signals were found in steroid-treated emus. SAON was found in all emus with a joint collapse incidence of 70%. The percentage of neutrophils (Neut %) and parameters on lipid metabolism significantly increased after induction. Micro-CT revealed structure deterioration of subchondral trabecular bone. Histomorphometry showed larger fat cell fraction and size, thinning of subchondral plate and cartilage layer, smaller osteoblast perimeter percentage and less blood vessels distributed at collapsed region in SAON group as compared with the normal controls. Scanning electron microscope (SEM) showed poor mineral matrix and more osteo-lacunae outline in the collapsed region in SAON group. The combination of pulsed LPS and MPS developed in the current study was safe and effective to induce SAON and deterioration of subchondral bone in bipedal emus with subsequent femoral head collapse, a typical clinical feature observed in patients under pulsed steroid treatment. In conclusion, bipedal emus could be used as an effective preclinical experimental model to evaluate potential treatment protocols to be developed for prevention of ON-induced hip joint collapse in patients

Spectroscopic Studies of Model Protein Interactions with Lipid Vesicles and Insulin Fibril Disassembly

The work in this thesis focuses on spectroscopic studies of refolding and interaction of model proteins with various lipid vesicles and on dimethyl sulfoxide (DMSO)-induced disassembly of various structural variants of insulin fibrils. The main spectroscopic methods utilized in this thesis include electronic circular dichroism (ECD), fluorescence, infrared absorption (IR) and vibrational circular dichroism (VCD). ECD, IR and VCD were used to characterize the secondary structure of model proteins based on their distinct bandshapes for various secondary structure elements. Polarized attenuated total reflectance (ATR)-FTIR was particularly useful in a protein-lipid membrane study (Chapter 3), because it can provide information about relative orientation of protein segments and the lipid bilayer. VCD was used to probe the disassembly of insulin fibrils (Chapter 4) due to its sensitivity to supramolecular chirality arising from higher-order self-assembly of aggregates. Fluorescence was used to monitor protein local tertiary structural change in protein-lipid vesicle interaction. In Chapter 3, a β-sheet to α-helix transformation of monomeric β-lactoglobulin (βLG) induced by small unilamellar vesicles (SUVs) of zwitterionic lipids at low pH was determined via various spectroscopic techniques. With SUVs of a zwitterionic lipid (1, 2-distearoyl-sn-glycero-3-phosphocholine, DSPC), βLG converted to a substantially helical form in a two-step kinetic process (fast and slow steps) monitored by CD. Fluorescence implied a rapid initial change in the Trp environments followed by a slower process paralleling the secondary structure change. Polarization ATR-FTIR results indicate the helices formed are at least partially inserted into the lipid bilayer and the sheet segments are on the surface. Thermal behavior showed that changes in the secondary structure for the lipid bound βLG occurred in three phases: the first is a slight reduction of the α-helix for βLG in the protein-lipid complex; the second is the DSPC phase change after which the proteins apparently dissociated from the vesicles and refolded into their native structures; the third is the unfolding of solvated βLG at high temperature. These thermal and kinetic behaviors suggest a different mechanism for the monomeric βLG interaction with zwitterionic lipids than was seen previously for the dimeric form at higher pH. In Chapter 4, VCD was utilized to characterize the macroscopic chirality and the DMSO-induced disassembly process for two types of insulin fibrils formed under different conditions. In this study it is confirmed that very high concentrations of DMSO both disaggregate these insulin fibrils and change their secondary structure. Inter-conversion of some insulin fibril types also occurred during the destabilization process as monitored by VCD. Transmission electron microscopy (TEM) images correlated the change in VCD sign pattern to alteration of morphology of the insulin fibrils. In Chapter 5, a computationally designed outer membrane protein (OmpFG) expected to form a monomeric β-barrel in the membrane was expressed and studied. At least partial refolding was evidenced in the lipid vesicles by CD detection of secondary structural change (random coil to β-sheet) and the change to a less polar environment of Trp residues was monitored by fluorescence. The results of a dye leakage assay indicate that the OmpFG can interact with lipid vesicles and may form a pore-like structure with relatively high conductivity

Quantitative analysis of correlation between AT and GC biases among bacterial genomes - Fig 4

<p><b>(A) Mean values of GC contents of genomes in each phylum. (B) Average percentages of genes in the leading strand grouped by genomes with the positive and negative ZCC indexes in each phylum.</b> In the histogram (A), mean values of GC content in N-ZCC phyla are entirely larger than those in P-ZCC phyla. The histogram (B) shows that genes are preferred to located in leading strands. Besides, the degree of strand-biased gene distribution (SGD) is generally stronger among genomes with positive ZCC indexes than those with negative ZCC indexes.</p

Summary information of ZCC indexes in different phyla.

<p>Summary information of ZCC indexes in different phyla.</p

Genome distributions to DE and PC groups in different phyla.

<p>Genome distributions to DE and PC groups in different phyla.</p

The Z-curve disparity figures.

<p>Among different genomes, GC disparity curves always show inverted-V curves, while the shapes of AT disparity curves vary from phyla, ZCC index signs and numerical values.</p

Equilibrium and Dynamic Spectroscopic Studies of the Interaction of Monomeric β‑Lactoglobulin with Lipid Vesicles at Low pH

β-Lactoglobulin (βLG) is a member of the lipocalin protein family that changes structure upon interacting with anionic surfactants and lipid vesicles under higher-pH conditions at which βLG is dimeric. In this study, a β-sheet to α-helix transformation was also observed for monomeric βLG obtained at pH 2.6 when it was mixed with small unilamellar vesicles (SUVs) of zwitterionic lipids, but being mixed with anionic lipids produced little change. The dynamics and extent of this change were quite dependent on the lipid character, phase, and vesicle size. With 1,2-distearoyl-<i>sn</i>-glycero-3-phosphocholine (DSPC), at ∼50 °C and pH 2.6, the βLG converted to a substantially helical form upon addition of ∼10 mM lipid in a two-step kinetic process having time constants of ∼1 and ∼25 h, as monitored by circular dichroism (CD). Fluorescence changes were simpler but implied a rapid initial change in the Trp environments followed by a slower process paralleling the change in secondary structure. Polarization attenuated total reflectance Fourier transform infrared results indicate the formed helices are at least partially inserted into the lipid bilayer and the sheet segments are on the surface. Thermal behavior showed that the secondary structure of the lipid-bound βLG had two phases, the first being characteristic of the protein–lipid vesicle interaction and the second following the DSPC phase change after which the protein apparently dissociated from the vesicle. Large unilamellar vesicles had a weaker interaction, as judged by CD, which may correlate to the partial exposure of the hydrophobic parts of the SUV bilayer. Other zwitterionic lipids bound βLG with much slower kinetics and often required sonication to induce interaction, but these also showed dissociation upon lipid phase change. These thermal and kinetic behaviors suggest a mechanism for the interaction of monomeric βLG with zwitterionic lipids different from that seen previously for the dimeric form

<i>In situ</i> single cell detection via microfluidic magnetic bead assay

<div><p>We present a single cell detection device based on magnetic bead assay and micro Coulter counters. This device consists of two successive micro Coulter counters, coupled with a high gradient magnetic field generated by an external magnet. The device can identify single cells in terms of the transit time difference of the cell through the two micro Coulter counters. Target cells are conjugated with magnetic beads via specific antibody and antigen binding. A target cell traveling through the two Coulter counters interacts with the magnetic field, and have a longer transit time at the 1st counter than that at the 2nd counter. In comparison, a non-target cell has no interaction with the magnetic field, and hence has nearly the same transit times through the two counters. Each cell passing through the two counters generates two consecutive voltage pulses one after the other; the pulse widths and magnitudes indicating the cell’s transit times through the counters and the cell’s size respectively. Thus, by measuring the pulse widths (transit times) of each cell through the two counters, each single target cell can be differentiated from non-target cells even if they have similar sizes. We experimentally proved that the target human umbilical vein endothelial cells (HUVECs) and non-target rat adipose-derived stem cells (rASCs) have significant different transit time distribution, from which we can determine the recognition regions for both cell groups quantitatively. We further demonstrated that within a mixed cell population of rASCs and HUVECs, HUVECs can be detected <i>in situ</i> and the measured HUVECs ratios agree well with the pre-set ratios. With the simple device structure and easy sample preparation, this method is expected to enable single cell detection in a continuous flow and can be applied to facilitate general cell detection applications such as stem cell identification and enumeration.</p></div

Microfluidic Magnetic Bead Assay for Cell Detection

We present a novel cell detection device based on a magnetic bead cell assay and microfluidic Coulter counting technology. The device cannot only accurately measure cells size distribution and concentration but also detect specific target cells. The device consists of two identical micro Coulter counters separated by a fluid chamber where an external magnetic field is applied. Antibody-functionalized magnetic beads were bound to specific antigens expressed on the target cells. A high-gradient magnetic field was applied to the chamber closer to the second counter via an external cylindrical magnet. Because of the magnetic interaction between the magnetic beads and the magnetic field, target cells were retarded by the magnetic field; transit time of a target cell (bound with magnetic beads) passing through the second counter was longer than that through the first counter. In comparison, transit times of a nontarget cell remained nearly the same when it passed through both counters. Thus, from the transit time delay we can identify target cells and quantify their concentration in a cell suspension. The transit time and the size of each cell were accurately measured in terms of the width and amplitude of the resistive pulses generated from the two Coulter counters. Experiments demonstrated that for mixed cells with various target cell ratios, the transit time delay increased approximately linearly with the increasing target cell ratio. The limit of detection (LOD) of the assay was estimated to be 5.6% in terms of target cell ratio. Cell viability tests further demonstrated that most cells were viable after the detection. With the simple device configuration and easy sample preparation, this rapid and reliable method is expected to accurately detect target cells and could be applied to facilitate stem cell isolation and characterization

Microscopic image of the HUVECs conjugated with magnetic beads.

<p>Microscopic image of the HUVECs conjugated with magnetic beads.</p